There is known an analytical method of comprehensively visualizing the expression amount of a protein expressed in tumor tissue based on signal strength using a time-of-flight secondary ion mass spectrometer (TOF-SIMS) (U.S. Pat. No. 7,446,309).
Moreover, the normalization of signal intensity of mass spectrometry using a standard material is known. In a proteomic analysis, proteins are separated by two-dimensional electrophoresis and the separated proteins are analyzed by mass spectroscopy. Then, the intensity of the protein of interest is normalized using the intensity of a protein having a known content as a standard to obtain a quantitative result.
A two-dimensional distribution image of a target protein can be formed based on the signal intensity of target proteins by using such as TOF-SIMS and matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). In such cases, there is known a method of normalizing the signal intensity using the total doses of primary ions, the irradiance of a pulse laser, or the resulting number of total counts of secondary ions as a standard.
The normalization using an external standard material is also proposed. Here, the external standard material refers to a standard material for normalizing the mass signal intensity obtained by mass spectroscopy, which is not contained in a measurement sample but added to the sample. The use of external standard material in TOF-SIMS is shown in Japanese Patent Application Laid-Open No. 2004-037120. In the publication, a material used as an external standard is arranged outside the measurement range of a sample. Then the signal intensity of the protein of interest in the sample is normalized by the signal intensity of the external standard. By this method, the signal intensities of the sample and the external standard can be obtained in a same condition.
The use of external standard material in MALDI-MS is also known. For example, it is known that a certain amount of specific peptide is mixed with a matrix agent and that the matrix is uniformly sprayed on a sample surface so as to use the peptide as an external standard to obtain a two-dimensional distribution image of the mass spectrum of constituents of a tissue section.
The normalization using an internal standard is also proposed. Here, the internal standard material refers to a standard material for normalizing the mass signal intensity obtained by mass spectroscopy, which is previously contained in a measurement sample. For example, in S. G. Ostrowski et al., Anal. Chem. 79, 3554 (2007), the signal intensity of cholesterol in a cell membrane surface is normalized using the signal intensity of the fragment ion obtained from a lipid component of the cell as a standard to form a two-dimensional distribution image by TOF-SIMS.